There is a considerable amount of prior art regarding the immobilization of E. coli or other microbial cells for use in the preparation of L-aspartic acid. For example, U.S. Pat. No. 3,791,926 (Chibata et al) describes a process for the production of L-aspartic acid which involves polymerizing a monomer selected from acrylamide, N,N'-lower alkylene-bis(acrylamide) and bis(acrylamidomethyl) either in an aqueous suspension containing an aspartase-producing microorganism such as E. coli ATCC no. 11303. The resultant immobilized aspartase-producing microorganism is treated with ammonium fumarate or a mixture of fumaric acid or its salt and an inorganic ammonium salt which by enzymatic reaction gives L-aspartic acid.
The immobilization of E. coli cells containing aspartase activity and use of the resulting immobilized cells for the production of L-aspartic acid are also described by Fusee et al, Applied and Environmental Microbiology, Vol. 42, No. 4, pages 672-676 (October 1981). According to Fusee et al, the cells are immobilized by mixing a suspension of the cells with a liquid isocyanate-capped polyurethane prepolymer (HYPOL.RTM.) so as to form a "foam" containing the immobilized cells.
Sato et al (Biochimica et Biophysica Acta, 570 (1979) pages 179-186) have disclosed the immobilization of E. coli cells containing aspartase activity with K-carrageenan, and use of the immobilized preparation for the production of L-aspartic acid.
Additional literature disclosures describing the immobilization of microbial cells in urethane prepolymers or polyurethanes or the like include the following:
(a) Immobilization of Microbial Cells in Polyurethane Matrices, by Klein et al, Biotechnology Letters, Vol. 3, No. 2, pages 65-70 (1981) PA1 (b) Hyrophilic Urethane Prepolymers: Convenient Materials for Enzyme Entrapment, Biotechnology and Bioengineering, Vol. XX, pages 1465-1469 (1978); PA1 (c) Transformation of Steroids by Gel-Entrapped Cells in Organic Solvent, by Omata et al, European J. Applied Microbiology and Biotechnology, 8, pages 143-155 (1979); and PA1 (d) Entrapment of Microbial Cells and Organelles With Hydrophilic Urethane Prepolymers, by Tanaka et al, European J. Applied Microbiology and Biotechnology, 7, 351-354 (1979). PA1 The provision of an improved process for preparing L-aspartic acid from ammonium fumarate using immobilized E. coli cells which maintain optimum L-aspartic activity for relatively long periods of time. PA1 The provision of an improved process for preparing L-phenylalanine from L-aspartic acid plus phenylpyruvate using immobilized E. coli cells which maintain optimum L-phenylalanine transaminase activity for relatively long periods of time. PA1 Molecular sieves PA1 Ion exchange resins PA1 Alumina PA1 Silica and silica gel PA1 Foraminifera skeletons PA1 Polymer latexes PA1 Metals PA1 Removal of part or nearly all of the water at temperatures below 60.degree. C. (usually between 40.degree. C. and 0.degree. C.) and pressures of 760 to 1.0 Torr. PA1 Allowing the composition to stand at temperatures below 60.degree. C. (usually between 40.degree.-0.degree. C.) without removing the water for periods of 0.5 to 50 hours. Compositions cured with the removal of water appear to give insoluble composites somewhat stronger than composites formed without the removal of water. PA1 Production of L-alanine via the immobilization of Pseudomonas dacunhae, or other microbe high in aspartase-.beta.-decarboxylase; PA1 Preparation of 6-amino-penicillanic acid via the immobilization of Bacillus megaterium, or other microbe high in penicillin acylase; PA1 High fructose corn syrup from immobilized Arthrobacter species ATCC 21748, or other microbes high in glucose isomerase, PA1 High fructose corn syrup from the glucose isomerase activity of immobilized Streptomyces phaeochromogenes; NRRL B3559; PA1 Production of prednisolone from the steriod dehydrogenase activity of immobilized Arthrobacter simplex; PA1 Production of L-phenylalanine from the phenylalanine ammonia lyase activity of immobilized Rhodosporidium toruloides ATCC 10788. PA1 (i) A batch type process wherein the catalyst compositions are stirred in from 0.1 to 5.0 molar (preferably 0.5 to 2.0 molar) solutions of ammonium fumarate in water at 5.0 to 10.0 pH (preferably 7.5 to 9.5 pH) for periods of 1.0 to 100 hours (preferably 8 to 48 hours) at temperatures below 60.degree. C. (preferably 20.degree. to 45.degree. C.). Broadly from 0.05 to 50 g of immobilized cells, preferably from 1.0 to 15 g, are used per 1.0 mole of starting ammonium fumarate. After the conversion, the catalyst compositions may be removed by filtration or the equivalent for reuse in converting fresh batches of fumarate solutions. The product solutions are obtained in a form suitable for conventional processing to isolate the L-aspartic acid (acidification, precipitation, filtration, washing, recrystallization, drying). PA1 (ii) A continuous process wherein the catalyst compositions, e.g. coated beads, are placed in columns and the solutions of ammonium fumarate (concentrations, pH's, and tmeperatures are the same as described above for the batch processes) are passed through the catalyst beds either from above or from below (fluidized bed mode). The rates of passage of these fumarate solutions may range from 0.1 to 1000 space velocities/hour. For example, 5.0 liters of solution per hour may be passed through 0.1 liter of catalyst bed representing 5.0 space velocities (S.V.) per hour. Preferably the fumarate solution flow rates which yield essentially 100% conversion of the fumarate to the L-aspartate fall in the range of 0.5 to 20.0 S.V./hour. The effluent from these columns of catalyst beds is suitable for conventional processing to isolate L-aspartic acid (as outlined above in the batch processes). PA1 (i) A batch type process wherein the catalyst compositions are stirred in from 0.01 to 0.5 molar (preferably 0.05 to 0.3 molar) solution of phenylpyruvate in the presence of 0.015 to 0.75 molar ammonium L-aspartate (preferably 0.075 to 0.5 molar) and 0.4 to 0.000001 mmolar pyridoxal 5-phosphate (preferably 0.04 to 0.0004 mmolar in water at 5.0 to 10.0 pH (preferably 7.5 to 9.5 pH) for periods of 1.0 to 100 hours (preferably 8 to 48 hours) at temperatures below 50.degree. C. (preferably 20.degree. to 40.degree. C.). Broadly from 0.05 to 50 g of immobilized cells, preferably from 1.0 to 15 g, are used per 0.1 mole of starting phenylpyruvate. After the conversion, the catalyst compositions may be removed by filtration or the equivalent for reuse in converting fresh batches of phenylpyruvate solutions. The product solutions are obtained in a form suitable for conventional processing to isolate the L-phenylalanine (ion exchange chromotography, precipitation, filtration, washing, recrystallization, drying). PA1 (ii) A continuous process wherein the catalyst compositions, e.g. coated beads, are placed in columns and the solutions of L-aspartate, phenylpyruvate and pyridoxal-5-phosphate (concentrations, pH's, and temperatures are the same as described above for the batch processes) are passed through the catalyst beds either from above or from below (fluidized bed mode). The rates of passage of these substrate solutions may range from 0.01 to 100 space velocities/hour. For example, 0.5 liters of solution per hour may be passed through 1.0 liter of catalyst bed representing 0.5 space velocities (S.V.) per hour. Preferably the phenylpyruvate/aspartate solution flow rates which yield essentially 100% conversion of the fumarate to the L-aspartate fall in the range of 0.05 to 0.5 S.V./hour. The effluent from these columns of catalyst beds is suitable for conventional processing to isolate L-phenylalanine (as outlined in the batch processes).
The above noted processes for preparing L-aspartic acid using immobilized microbial cells suffer from various disadvantages. For example, K-carrageenan gum and polyurethane "foam" as disclosed by Fusee et al and Sato et al are relatively soft and compressible. Hence when these immobilized cell compositions are used, in a column through which ammonium fumarate is passed for conversion to ammonium aspartase, they tend to be compressed and plug up, particularly where high flow rates and/or relatively tall column heights are involved.